EP0296136A1 - Biospecific multianalyte assay method with labelled microparticles - Google Patents

Biospecific multianalyte assay method with labelled microparticles Download PDF

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Publication number
EP0296136A1
EP0296136A1 EP88850205A EP88850205A EP0296136A1 EP 0296136 A1 EP0296136 A1 EP 0296136A1 EP 88850205 A EP88850205 A EP 88850205A EP 88850205 A EP88850205 A EP 88850205A EP 0296136 A1 EP0296136 A1 EP 0296136A1
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decay time
microspheres
biospecific
microsphere
suspension
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EP0296136B1 (en
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Erkki Soini
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Wallac Oy
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Wallac Oy
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers

Definitions

  • the invention relates to a biospecific multianalyte assay method.
  • Biocomplementary assays are a well established group of biospecific assays and now widely used in routine diagnostics and research laboratories. Another group of biospecific assays, still being developed, is DNA hybridization assays. Bio­specific assays generally employ one or several biospecific reactants (e.g. antibody, DNA probe) and normally one of these reactants is labelled. The labels currently used are radioisotopic, enzymatic, luminescent and fluorescent labels.
  • the purpose of this invention is to improve the methodology for biospecific multianalyte assays.
  • the method according to the invention is characterized by preparing categories of microspheres representing different analytes to be assayed, said categories comprising different amounts of a fluorescent substance having a short decay time, coating each category of microspheres with a biospecific reactant, pooling the different categories of microspheres together in a suspension, adding a sample containing analytes to be assayed to the suspension, adding a mixture of biospecific reactants labelled with a fluorescent compound having a long decay time to the suspension to initiate biospecific reactions between the analytes and the labelled reactants and microsphere-­associated reactants, diluting the suspension to reduce the concentration of labelled reactants not bound to the microspheres, exciting both the fluorescent substance having a short decay time and the fluorescent compound having a long decay time, associated with the microspheres, to generate fluorescence emissions, converting the fluorescence emissions to electrical signals, identifying the category of each microsphere on the basis of the strength of the electrical signal resulting from the short decay time fluorescent substance, measuring the concentration of the analy
  • the invention combines the use of microspheres and fluore­scent labels with substantially different fluorescence decay times.
  • the multianalyte assay is performed in a suspension of artificially manufactured microspheres, comprising a pool of different microspheres representing different analytes, here called categories.
  • Each category of microspheres is first coated with a specific reactant (antibody), i.e. the microspheres function as solid supports for said specific reactant and for the biospecific reaction.
  • This invention is particularly related to a new microfluoro­metric methodology combining short decay time and long decay time fluorescent labels where short decay time labels are used for identification of the category of each individual microsphere representing different analytes and the long decay time labels are used for detecting the concentration of the particular analyte on the microsphere by means of a biospecific reaction.
  • Monosized microspheres can be made of appropriate polymer material with slight hydrophilic character and therefore are well suited for water suspensions.
  • the microsphere surface properties allow also binding of macromolecules by physical adsorption as well as by covalent coupling through activation of OH-groups at the surface (L. Johansen & al., Journal of Immunological Methods, 59 (1983) pp. 255-264).
  • the sample including all different analytes is first incubated with a pool of micro­spheres and with a pool of labelled reactants in smallest possible volume (for example 10 - 100 ⁇ l) in order to achieve a complete reaction in a short time.
  • the equilibrium of the biospecific reaction is achieved rapidly during the incubation and as a result conjugates consisting of the labelled reactant, the analyte and the immobilized reactant on the microsphere surface are formed, commonly called “sandwiches" in the literature.
  • the suspension is diluted adequately for analyzing the individual microspheres fluorometrically. A sufficient number of microspheres are analyzed and the fluorescence signals from each microsphere are registered in a computer.
  • high detection sensitivity of the fluorescent labels associated with the biospecific reactants at the microparticle surface is based on the use of long decay time fluorescent labels e.g. lanthanide chelates of europium, terbium etc as described in more details for example in the European patent application 86850172.7.
  • the detection sensitivity is improved by reducing the back­ground fluorescence with temporal resolution between the excitation and the emission which is usually called time-­resolved fluorescence detection in the literature.
  • a device for time-resolved fluorescence detection typically incorporates a pulsed light source and a gated detector, which is activated after a certain delay time from the excitation pulse.
  • a gated detector which is activated after a certain delay time from the excitation pulse.
  • the detector detects only long decay time emission which is specific to the long decay time fluore­scent label in the sample.
  • the system relative to this invention combines the features referred to above and the objective of this invention is to identify each individual microparticle representing different analyte categories for the purpose to perform a multianalyte assay in the same sample.
  • the identification is based on detection of short decay time fluorescence as discussed above.
  • the short decay time and the long decay time fluorescence can be separately detected.
  • a strong and variable short decay time fluorescence component makes negligible contribution to the detection of the long decay fluorescence component. This is the crucial point of this invention and the fast decaying fluorescent material can be used for identification of the categories of the microparticles without any significant interference to the determination of the analyte concentration.
  • Microspheres can be manufactured combining the polymer material with a suitable short decay time fluorescent compound.
  • Organic fluorescent compounds with very short decay time for example POPOP, bisMSB etc.
  • POPOP photosensitive polymer
  • bisMSB organic fluorescent compounds with very short decay time
  • any monomer as discussed e.g. in "Theory and practice of Scintillation Counting", Ed. J.B. Birks, Pergamon Press, 1967, pp. 321-353
  • solid fluorescent material is formed in polymerization.
  • the material is processed into micro­spheres in the same step.
  • the short decay time organic fluorescent compounds are added into different batches of the monomer in substantially different concentrations differing e.g. by a factor of two from each other.
  • the identification signal is detected simultaneously with the excitation which lasts for at the most some micro­seconds and is substantially shorter than the decay time of the long decay time fluorescent label.
  • the contribution of the long decay time fluorescence label to the identi­fication signal level is very low because the photon emission rate of the long decay time component per micro­second is very low and because the identification compound can be used in much higher concentrations than the long decay time label.
  • the cytometer simultaneously measured fluorescent light and scattered light from the microspheres, it was possible to measure only the microsphere-associated fluorescence. Consequently the special advantage of this methodology was that no macroscopic separation of bound and free labelled compounds were needed. Macroscopic separation was circumvented by the ability of the flow cytometer to discriminate, at the microscopic level, particle associated (bound) fluorescence from solution-­associated (free) fluorescence. The discrimination was most efficient for 1-5 ⁇ m diameter microspheres, where spheres in a highly fluorescent background medium and spheres in a buffer medium displayed approximately the same fluorescent intensity per particle.
  • the concept of temporal resolution between short decay time fluorescence and long decay time fluorescence is, however, not based on the use of the gated detector as described above.
  • the temporal resolution between the excitation and emission can be achieved by high speed movement of the sample in relation to the focal points of excitation and detection.
  • the focal points of excitation and emission are separated by a small distance in such a way that the signals from scattering and short decay time fluorescence are not detected but the signals from any long decay time fluorescent label can be detected, because of their "after glow" when the sample moves at a high speed in relation to the focal points.
  • An apparatus incorporating the features discussed above, can be constructed for example as illustrated in Fig. 1.
  • an europium chelate is used as a long decay time label and POPOP is used as a short decay time fluorescent substance, i.e. identification substance.
  • the excitation wavelengths of europium chelates and POPOP are generally in the same range between 300 nm and 360 nm.
  • the 337 nm wavelength emission from a nitrogen laser or from a xenon flash lamp is optimal for excitation.
  • Emission wavelengths are 613 nm for europium and around 400 nm for POPOP.
  • the excitation and emission spectra are shown in Fig. 2.
  • a suspension of microparticles 1 and a sheath fluid 2 are moving through a sheath flow nozzle 3 into a thin laminar flow 4, which is first illuminated with a light beam 5 from a continuous light source 6, for example a HeNe-laser at a wavelength of 633 nm, focussed to a point 8 in the laminar flow 4.
  • a photon detector 7 sensitive for 633 nm is focussed to the point 8 and used for monitoring the photon scattering from randomly incoming microparticles 1.
  • Each microparticle 1 produces a signal in the detector 7 and this signal is used for triggering a flash lamp or a pulsed laser 9 which is focussed to a point 10 in the laminar flow 4.
  • the pulsed light source 9 is used for excitation of both fluorescent substances, i.e. the identification compound and the lanthanide chelate in the microparticles at a wavelength of 337 nm.
  • the signal from the detector 7 also activates a computer 11.
  • a gated photon detector 12 sensitive for a wavelength of 400 nm is focussed to the point 10 and measures the amplitude of the short decay time signal from the organic fluorescent compound of each microparticle 1. This amplitude measure­ ment is then used for identification of the particle by the computer 11.
  • a detector 13 measures the long decay time fluorescence at a wavelength of 614 nm. The detector 13 is focussed to a point 14 at a small distance dx down­stream from the point 10.
  • the small distance dx v ⁇ t d , where v is the speed of the laminar flow 4 and t d is a delay time.
  • a gate 15 is activated for a counting time t g after the delay time t d .
  • the delay time t d and the counting time t g are controlled by the computer 11 and are optimized for highest signal-to-background ratio and depend on the decay time of the long decay time fluorescent label.
  • the counting speed of the system depends on the maximum pulse frequency of the pulsed light source 9 and on the counting time t g of the signal from the long decay time label. Sig­nals from microparticles which coincide within a time inter­val shorter than the minimum light pulse period, can be electronically rejected.
  • the computer 11 integrates photon signals from microparticles separately for each parameter. Using appropriate standard samples of each analyte, the system can be calibrated for concentration units.
  • the flow cytometric systems are relatively expensive and their optical and hydrodynamic systems are mechanically complex and sensitive.
  • the analysis of the micro­particles can also be performed in a stationary state or in a stopped-flow system and this type of approach may be less expensive and more robust.
  • the stationary instru­mental approach is based on the use of microscopic observations of microparticles in a thin cavity into which the diluted microparticle suspension is pumped from a reaction tube. The suspension of the microparticles is first scanned at a very rapid rate and the identification of the microspheres as well as their location and determin­ation of their analyte categories take place on the basis of the detection of short decay time fluorescence.
  • the detection of the analyte concentration in the micro­particles takes place measuring the signal strength of long decay time fluorescence. A sufficient number of randomly located microparticles are identified and analyzed in order to obtain a sufficient amount of informa­tion for desired statistical precision.
  • the excitation light source can be either a pulsed light source or a constant light source. If using a pulsed light source, each scanned point of the sample is exposed to one or several light pulses and the detector and the associated photon counting electronics is gated for detection of both short decay time and long decay time fluorescence separately.
  • the concept of temporal resolu­tion between short decay time fluorescence and long decay time fluorescence can be achieved in the following manner.
  • the focal points of the excitation and the emission are separated by a small distance in such a way that the signals from the short decay time fluorescence and the signals from the long decay time fluorescent label can be detected at different times when the excitation beam moves at a high speed in relation to the sample. This concept is discussed in more detail in the Swedish patent applica­tion 8604434-4.
  • Microfluorometric analysis (as well as flow cytometric) can discriminate between the microsphere-associated fluorescence and solution-associated fluorescence, because the respective concentrations in the microscopic focal point in the diluted suspension differ many orders of magni­tude. This detection concept provides a reasonable separation of the free and the bound fractions but if necessary, it can be made more efficient using normal separation methods (washing, filtration, centrifugation etc.)
  • Detection of various fluorescence signals can be made with an appropriate computer controlled automatic video micro­scope (Video Microscopy, ed. Shinya Inoue, Plenum Press, 1986) or with a scanning microscope.
  • the scanning microscope can also be equipped with confocal optical systems for excitation and emission in order to improve sensitivity and precision. (G.J. Brakenhof & al., J. Microscopy, 117 (1979) pp. 219-232).
  • the aperture can be selected optimally according to the diameter of the micro­particles and scanning of the fluorescence emission signals can be made using an appropriate imaging detector which can work in a single photon mode.
  • Such detectors can be composed for example of microchannel plate image intensifiers and charge coupled imaging devices (CCD).
  • Charge coupled devices can be gated for the purpose of time resolved operation in order to discriminate between short decay time fluorescence and long decay time fluores­cence (see Video Microscopy, ed Shinya Inoue'). Analysis of the image from the CCD can be performed with a computer.

Abstract

In a biospecific multianalyte assay the use of micro­spheres and fluorescent labels with substantially different fluorescence decay times, is combined. The assay is performed in a suspension of microspheres in the form of a pool of different microsphere categories, where the categories represent different analytes. The microspheres belonging to the respective categories are first coated with a specific reactant, i.e. the micro­spheres function as a solid support for the reactant and for a biospecific reaction. Fluorescent labels having a short decay time are used to identify the category of each individual microsphere, while fluorescent labels having a long decay time are used to determine the con­centration of a particular analyte on the microsphere by means of the biospecific reaction.

Description

  • The invention relates to a biospecific multianalyte assay method.
  • Immunoassays are a well established group of biospecific assays and now widely used in routine diagnostics and research laboratories. Another group of biospecific assays, still being developed, is DNA hybridization assays. Bio­specific assays generally employ one or several biospecific reactants (e.g. antibody, DNA probe) and normally one of these reactants is labelled. The labels currently used are radioisotopic, enzymatic, luminescent and fluorescent labels.
  • In routine diagnostics there is a growing need for multi­analyte (multiparameter) analysis. Unfortunately, the present methodology does not allow the use of more than two or three simultaneous labels because the spectrometric separation of the signals from different labels is not sufficiently efficient. The emission spectra of different radioisotopic labels and fluorometric labels have a significant overlapping and consequently they provide in­adequate separation of different analytes over a required concentration range.
  • The purpose of this invention is to improve the methodology for biospecific multianalyte assays.
  • The method according to the invention is characterized by preparing categories of microspheres representing different analytes to be assayed, said categories comprising different amounts of a fluorescent substance having a short decay time, coating each category of microspheres with a biospecific reactant,
    pooling the different categories of microspheres together in a suspension,
    adding a sample containing analytes to be assayed to the suspension,
    adding a mixture of biospecific reactants labelled with a fluorescent compound having a long decay time to the suspension to initiate biospecific reactions between the analytes and the labelled reactants and microsphere-­associated reactants,
    diluting the suspension to reduce the concentration of labelled reactants not bound to the microspheres,
    exciting both the fluorescent substance having a short decay time and the fluorescent compound having a long decay time, associated with the microspheres, to generate fluorescence emissions,
    converting the fluorescence emissions to electrical signals, identifying the category of each microsphere on the basis of the strength of the electrical signal resulting from the short decay time fluorescent substance,
    measuring the concentration of the analyte on each micro­sphere on the basis of the strength of the electrical signal resulting from the long decay time fluorescent compound.
  • The invention will be described more in detail with reference to the accompanying drawings on which
    • Fig. 1 schematically shows an embodiment of an apparatus for carrying the method according to the invention into effect, and
    • Fig. 2 shows excitation and emission spectra.
  • The invention combines the use of microspheres and fluore­scent labels with substantially different fluorescence decay times. The multianalyte assay is performed in a suspension of artificially manufactured microspheres, comprising a pool of different microspheres representing different analytes, here called categories. Each category of microspheres is first coated with a specific reactant (antibody), i.e. the microspheres function as solid supports for said specific reactant and for the biospecific reaction. This invention is particularly related to a new microfluoro­metric methodology combining short decay time and long decay time fluorescent labels where short decay time labels are used for identification of the category of each individual microsphere representing different analytes and the long decay time labels are used for detecting the concentration of the particular analyte on the microsphere by means of a biospecific reaction.
  • Monosized microspheres can be made of appropriate polymer material with slight hydrophilic character and therefore are well suited for water suspensions. The microsphere surface properties allow also binding of macromolecules by physical adsorption as well as by covalent coupling through activation of OH-groups at the surface (L. Johansen & al., Journal of Immunological Methods, 59 (1983) pp. 255-264). In this invention the sample including all different analytes is first incubated with a pool of micro­spheres and with a pool of labelled reactants in smallest possible volume (for example 10 - 100 µl) in order to achieve a complete reaction in a short time. Because of the very small average distance between the analyte molecules and labelled reactants in the microsphere suspen­sion, the equilibrium of the biospecific reaction is achieved rapidly during the incubation and as a result conjugates consisting of the labelled reactant, the analyte and the immobilized reactant on the microsphere surface are formed, commonly called "sandwiches" in the literature. After the incubation the suspension is diluted adequately for analyzing the individual microspheres fluorometrically. A sufficient number of microspheres are analyzed and the fluorescence signals from each microsphere are registered in a computer.
  • In this invention high detection sensitivity of the fluorescent labels associated with the biospecific reactants at the microparticle surface is based on the use of long decay time fluorescent labels e.g. lanthanide chelates of europium, terbium etc as described in more details for example in the European patent application 86850172.7. The detection sensitivity is improved by reducing the back­ground fluorescence with temporal resolution between the excitation and the emission which is usually called time-­resolved fluorescence detection in the literature. A comprehensive review of the most recent developments and the evolution of the time-resolved fluorometry of long decay time fluorescent labels has been written by Soini and Lövgren(CRC Critical Reviews in Analytical Chemistry,Vol. 18, Issue 2(1987)).A device for time-resolved fluorescence detection typically incorporates a pulsed light source and a gated detector, which is activated after a certain delay time from the excitation pulse. Thus the short decay time fluorescence or the scattering is not registered by the detector because its lifetime is much shorter than the time delay. The detector detects only long decay time emission which is specific to the long decay time fluore­scent label in the sample.
  • The system relative to this invention combines the features referred to above and the objective of this invention is to identify each individual microparticle representing different analyte categories for the purpose to perform a multianalyte assay in the same sample. The identification is based on detection of short decay time fluorescence as discussed above. In an appropriate measuring system, the short decay time and the long decay time fluorescence can be separately detected. In particular, when the decay times differ several orders of magnitude (organic fluore­scent material in the nanosecond range and lanthanide chelates in the microsecond to millisecond range), a strong and variable short decay time fluorescence component makes negligible contribution to the detection of the long decay fluorescence component. This is the crucial point of this invention and the fast decaying fluorescent material can be used for identification of the categories of the microparticles without any significant interference to the determination of the analyte concentration.
  • Microspheres can be manufactured combining the polymer material with a suitable short decay time fluorescent compound. Organic fluorescent compounds with very short decay time, for example POPOP, bisMSB etc., can be added to any monomer (as discussed e.g. in "Theory and practice of Scintillation Counting", Ed. J.B. Birks, Pergamon Press, 1967, pp. 321-353) and solid fluorescent material is formed in polymerization. The material is processed into micro­spheres in the same step. For identification of the micro­spheres, the short decay time organic fluorescent compounds are added into different batches of the monomer in substantially different concentrations differing e.g. by a factor of two from each other.
  • The identification signal is detected simultaneously with the excitation which lasts for at the most some micro­seconds and is substantially shorter than the decay time of the long decay time fluorescent label. The contribution of the long decay time fluorescence label to the identi­fication signal level is very low because the photon emission rate of the long decay time component per micro­second is very low and because the identification compound can be used in much higher concentrations than the long decay time label.
    • The use of flow cytometers as analysers
  • It is known that flow cytometers (J. Steinkamp, Rev.Sci. Instrum. 55 (1984) pp. 1375-1400) can be used for immuno­assays employing microspheres as a solid support (P.J. Lisi & al., Clinica Chimica Acta, 120 (1982) pp. 171-179). In this method antigen-coated or antibody-coated microspheres (1-50 µm diameter) were allowed to react with the sample and with fluorescent-labelled antigen or antibody in any generally employed immunoassay format, i.e. competitive binding, double antibody "sandwich", etc. The suspension was introduced unwashed into a flow cytometer and the microspheres flowed in a narrow stream past a laser beam.
  • Provided that the cytometer simultaneously measured fluorescent light and scattered light from the microspheres, it was possible to measure only the microsphere-associated fluorescence. Consequently the special advantage of this methodology was that no macroscopic separation of bound and free labelled compounds were needed. Macroscopic separation was circumvented by the ability of the flow cytometer to discriminate, at the microscopic level, particle associated (bound) fluorescence from solution-­associated (free) fluorescence. The discrimination was most efficient for 1-5 µm diameter microspheres, where spheres in a highly fluorescent background medium and spheres in a buffer medium displayed approximately the same fluorescent intensity per particle.
  • A possibility of using flow cytometers for multianalyte immunoassay has been discussed by T.M.McHugh & al., J. Immunol. Methods 95 (1986) pp 57-61 where microspheres of different sizes were used as solid support and the identification of microspheres associated with different analytes was based on analysing the sizes of microspheres. This has been found feasible, because the flow cytometer can accurately detect particles based on size and it would be possible to identify a known microsphere population and to determine the fluorescence without interference from other size microspheres.
  • In a flow cytometer the concept of temporal resolution between short decay time fluorescence and long decay time fluorescence is, however, not based on the use of the gated detector as described above. The temporal resolution between the excitation and emission can be achieved by high speed movement of the sample in relation to the focal points of excitation and detection. In this concept, as discussed in more details in the Swedish patent application 8604434-4, the focal points of excitation and emission are separated by a small distance in such a way that the signals from scattering and short decay time fluorescence are not detected but the signals from any long decay time fluorescent label can be detected, because of their "after glow" when the sample moves at a high speed in relation to the focal points.
  • An apparatus incorporating the features discussed above, can be constructed for example as illustrated in Fig. 1. In this example it has been supposed that an europium chelate is used as a long decay time label and POPOP is used as a short decay time fluorescent substance, i.e. identification substance. The excitation wavelengths of europium chelates and POPOP are generally in the same range between 300 nm and 360 nm. The 337 nm wavelength emission from a nitrogen laser or from a xenon flash lamp is optimal for excitation. Emission wavelengths are 613 nm for europium and around 400 nm for POPOP. The excitation and emission spectra are shown in Fig. 2.
  • A suspension of microparticles 1 and a sheath fluid 2 are moving through a sheath flow nozzle 3 into a thin laminar flow 4, which is first illuminated with a light beam 5 from a continuous light source 6, for example a HeNe-laser at a wavelength of 633 nm, focussed to a point 8 in the laminar flow 4. A photon detector 7 sensitive for 633 nm is focussed to the point 8 and used for monitoring the photon scattering from randomly incoming microparticles 1. Each microparticle 1 produces a signal in the detector 7 and this signal is used for triggering a flash lamp or a pulsed laser 9 which is focussed to a point 10 in the laminar flow 4. The pulsed light source 9 is used for excitation of both fluorescent substances, i.e. the identification compound and the lanthanide chelate in the microparticles at a wavelength of 337 nm. The signal from the detector 7 also activates a computer 11. A gated photon detector 12 sensitive for a wavelength of 400 nm is focussed to the point 10 and measures the amplitude of the short decay time signal from the organic fluorescent compound of each microparticle 1. This amplitude measure­ ment is then used for identification of the particle by the computer 11. A detector 13 measures the long decay time fluorescence at a wavelength of 614 nm. The detector 13 is focussed to a point 14 at a small distance dx down­stream from the point 10. The small distance dx = v · td, where v is the speed of the laminar flow 4 and td is a delay time. A gate 15 is activated for a counting time tg after the delay time td. The delay time td and the counting time tg are controlled by the computer 11 and are optimized for highest signal-to-background ratio and depend on the decay time of the long decay time fluorescent label. The counting speed of the system depends on the maximum pulse frequency of the pulsed light source 9 and on the counting time tg of the signal from the long decay time label. Sig­nals from microparticles which coincide within a time inter­val shorter than the minimum light pulse period, can be electronically rejected. The computer 11 integrates photon signals from microparticles separately for each parameter. Using appropriate standard samples of each analyte, the system can be calibrated for concentration units.
  • The choice of long decay time fluorescent label affects the system parameters of the device. A special care should be taken for the leakage of stray light from the light source 6 to the detector 13. Good optics for focussing, optical separation of the points 8 and 14 as well as gating the detectors reduce the background caused by this effect.
    • The use of stationary microfluorometric analysers.
  • The flow cytometric systems are relatively expensive and their optical and hydrodynamic systems are mechanically complex and sensitive. However, the analysis of the micro­particles can also be performed in a stationary state or in a stopped-flow system and this type of approach may be less expensive and more robust. The stationary instru­mental approach is based on the use of microscopic observations of microparticles in a thin cavity into which the diluted microparticle suspension is pumped from a reaction tube. The suspension of the microparticles is first scanned at a very rapid rate and the identification of the microspheres as well as their location and determin­ation of their analyte categories take place on the basis of the detection of short decay time fluorescence. The detection of the analyte concentration in the micro­particles takes place measuring the signal strength of long decay time fluorescence. A sufficient number of randomly located microparticles are identified and analyzed in order to obtain a sufficient amount of informa­tion for desired statistical precision.
  • The excitation light source can be either a pulsed light source or a constant light source. If using a pulsed light source, each scanned point of the sample is exposed to one or several light pulses and the detector and the associated photon counting electronics is gated for detection of both short decay time and long decay time fluorescence separately.
  • If the constant light source is used in a confocal scanning microfluorometry, the concept of temporal resolu­tion between short decay time fluorescence and long decay time fluorescence can be achieved in the following manner. The focal points of the excitation and the emission are separated by a small distance in such a way that the signals from the short decay time fluorescence and the signals from the long decay time fluorescent label can be detected at different times when the excitation beam moves at a high speed in relation to the sample. This concept is discussed in more detail in the Swedish patent applica­tion 8604434-4.
  • Microfluorometric analysis (as well as flow cytometric) can discriminate between the microsphere-associated fluorescence and solution-associated fluorescence, because the respective concentrations in the microscopic focal point in the diluted suspension differ many orders of magni­tude. This detection concept provides a reasonable separation of the free and the bound fractions but if necessary, it can be made more efficient using normal separation methods (washing, filtration, centrifugation etc.)
  • Detection of various fluorescence signals can be made with an appropriate computer controlled automatic video micro­scope (Video Microscopy, ed. Shinya Inoue, Plenum Press, 1986) or with a scanning microscope. The scanning microscope can also be equipped with confocal optical systems for excitation and emission in order to improve sensitivity and precision. (G.J. Brakenhof & al., J. Microscopy, 117 (1979) pp. 219-232). Alternatively the aperture can be selected optimally according to the diameter of the micro­particles and scanning of the fluorescence emission signals can be made using an appropriate imaging detector which can work in a single photon mode. Such detectors can be composed for example of microchannel plate image intensifiers and charge coupled imaging devices (CCD). Charge coupled devices can be gated for the purpose of time resolved operation in order to discriminate between short decay time fluorescence and long decay time fluores­cence (see Video Microscopy, ed Shinya Inoue'). Analysis of the image from the CCD can be performed with a computer.

Claims (1)

  1. Biospecific multianalyte assay method, characterized by preparing categories of microspheres representing different analytes to be assayed, said categories comprising different amounts of a fluorescent substance having a short decay time, coating each category of microspheres with a biospecific reactant,
    pooling the different categories of microspheres together in a suspension,
    adding a sample containing analytes to be assayed to the suspension,
    adding a mixture of biospecific reactants labelled with a fluorescent compound having a long decay time to the suspension to initiate biospecific reactions between the analytes and the labelled reactants and microsphere-­associated reactants,
    diluting the suspension to reduce the concentration of labelled reactants not bound to the microspheres,
    exciting both the fluorescent substance having a short decay time and the fluorescent compound having a long decay time, associated with the microspheres, to generate fluorescence emissions,
    converting the fluorescence emissions to electrical signals, identifying the category of each microsphere on the basis of the strength of the electrical signal resulting from the short decay time fluorescent substance,
    measuring the concentration of the analyte on each microsphere on the basis of the strength of the electrical signal resulting from the long decay time fluorescent compound.
EP88850205A 1987-06-16 1988-06-07 Biospecific multianalyte assay method with labelled microparticles Expired EP0296136B1 (en)

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SE8702511 1987-06-16
SE8702511A SE458968B (en) 1987-06-16 1987-06-16 BIOSPECIFIC ANALYTICAL PROCEDURE FOR MULTIPLE ANALYTICS WHICH DO NOT INCLUDE PARTICULAR COATING AND LABELING WITH FLUORESCING LABEL SUBSTANCES

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EP0296136A1 true EP0296136A1 (en) 1988-12-21
EP0296136B1 EP0296136B1 (en) 1992-04-22

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991012530A1 (en) * 1990-02-16 1991-08-22 Pa Consulting Services Limited Radiative transfer fluorescence assay
WO1993003379A1 (en) * 1991-07-26 1993-02-18 E.I. Du Pont De Nemours And Company An assay with signal detection in the presence of a suspended solid support
EP0531933A2 (en) * 1991-09-09 1993-03-17 Canon Kabushiki Kaisha Quantitative determination of liquids, pure or complexed with other components
EP0563998A1 (en) * 1992-04-02 1993-10-06 Markus Sauer A process for detecting biomolecules, toxic substances, polymers and pharmaceutical substances by time-resolved laser spectroscopy
WO1994001774A1 (en) * 1992-07-02 1994-01-20 Erkki Soini Biospecific multiparameter assay method
EP0617286A2 (en) * 1993-03-18 1994-09-28 Wallac Oy Biospecific solid phase carrier
EP0617287A3 (en) * 1993-03-16 1995-07-26 Wallac Oy Biospecific assay method.
WO1996031777A1 (en) * 1995-04-03 1996-10-10 Macquarie Research Limited Method for detecting microorganisms
EP0749012A1 (en) * 1995-06-15 1996-12-18 Institut Français du Pétrole Device and method for determining the temporal behaviour of a transient phenomenon
US5702887A (en) * 1993-03-19 1997-12-30 Chiron Diagnostics Corporation Long emission wavelength chemiluminescent compounds and their use in test assays
FR2768519A1 (en) * 1997-09-18 1999-03-19 Immunotech Sa Standardisation of specific binding assays
EP0446245B1 (en) * 1988-11-03 1999-05-12 Igen International, Inc. Electrochemiluminescent assays
WO2000012123A2 (en) * 1998-08-28 2000-03-09 Febit Ferrarius Biotechnology Gmbh Method and measuring device for determining a plurality of analytes in a sample
EP1121582A2 (en) * 1998-08-21 2001-08-08 SurroMed, Inc. Novel optical architectures for microvolume laser-scanning cytometers
US6342397B1 (en) * 1998-06-04 2002-01-29 Erkki Soini Homogeneous biospecific assay using a solid phase, two-photon excitation and confocal fluorescence detection
WO2002027300A1 (en) * 2000-09-27 2002-04-04 Amersham Biosciences Uk Ltd Measurement of fluorescence decay times
WO2002061427A1 (en) * 2001-01-31 2002-08-08 Novozymes A/S Method of analysing granular composition by fluorescence analysis
WO2004106931A1 (en) * 2003-05-26 2004-12-09 Institut Virion\Serion Gmbh Method and testing system for analyzing and/or detecting biomolecules and/or active substances in liquid samples
EP1872128A2 (en) * 2005-04-20 2008-01-02 Becton, Dickinson and Company Multiplex microparticle system
WO2016156503A1 (en) * 2015-04-01 2016-10-06 Spx Flow Technology Danmark A/S A method and a system for monitoring spray nozzles in a spray drying or spray cooling chamber

Families Citing this family (148)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6379895B1 (en) 1989-06-07 2002-04-30 Affymetrix, Inc. Photolithographic and other means for manufacturing arrays
US6551784B2 (en) 1989-06-07 2003-04-22 Affymetrix Inc Method of comparing nucleic acid sequences
US6040138A (en) 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US5143854A (en) 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US5744101A (en) 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US5800992A (en) 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids
US5547839A (en) 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
DE69132843T2 (en) * 1990-12-06 2002-09-12 Affymetrix Inc N D Ges D Staat Identification of nucleic acids in samples
WO1993008472A1 (en) * 1991-10-15 1993-04-29 Multilyte Limited Binding assay employing labelled reagent
FI925064A (en) * 1992-11-09 1994-05-10 Erkki Juhani Soini Methods and apparatus for bioaffinicity testing
US7378236B1 (en) 1994-06-17 2008-05-27 The Board Of Trustees Of The Leland Stanford Junior University Method for analyzing gene expression patterns
US5807522A (en) * 1994-06-17 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods for fabricating microarrays of biological samples
US7323298B1 (en) 1994-06-17 2008-01-29 The Board Of Trustees Of The Leland Stanford Junior University Microarray for determining the relative abundances of polynuceotide sequences
USRE43097E1 (en) 1994-10-13 2012-01-10 Illumina, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US5604097A (en) 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
US8236493B2 (en) * 1994-10-21 2012-08-07 Affymetrix, Inc. Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA
FI98765C (en) 1995-01-16 1997-08-11 Erkki Soini Flow cytometric method and apparatus
JP3215428B2 (en) * 1995-01-16 2001-10-09 ソイニ，エルッキ Biospecific multi-parameter assays
FI101829B (en) * 1995-03-07 1998-08-31 Erkki Juhani Soini Biospecific assay method
EP0852004B1 (en) * 1995-10-11 2011-01-19 Luminex Corporation Multiplexed analysis of clinical specimens
US5981180A (en) * 1995-10-11 1999-11-09 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and methods
US5736330A (en) * 1995-10-11 1998-04-07 Luminex Corporation Method and compositions for flow cytometric determination of DNA sequences
EP0880598A4 (en) * 1996-01-23 2005-02-23 Affymetrix Inc Nucleic acid analysis techniques
US6387707B1 (en) * 1996-04-25 2002-05-14 Bioarray Solutions Array Cytometry
US6958245B2 (en) 1996-04-25 2005-10-25 Bioarray Solutions Ltd. Array cytometry
US7041510B2 (en) 1996-04-25 2006-05-09 Bioarray Solutions Ltd. System and method for programmable illumination pattern generation
US7144119B2 (en) * 1996-04-25 2006-12-05 Bioarray Solutions Ltd. System and method for programmable illumination pattern generation
EP2290364A1 (en) 1996-04-25 2011-03-02 BioArray Solutions Ltd. Light-controlled electrokinetic assembly of particles near surfaces
DE19634873A1 (en) * 1996-08-29 1998-03-12 Boehringer Mannheim Gmbh System for the differentiation of fluorescent groups of molecules by time-resolved fluorescence measurement
US6449562B1 (en) 1996-10-10 2002-09-10 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
US6327410B1 (en) * 1997-03-14 2001-12-04 The Trustees Of Tufts College Target analyte sensors utilizing Microspheres
US6023540A (en) 1997-03-14 2000-02-08 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
US20030027126A1 (en) * 1997-03-14 2003-02-06 Walt David R. Methods for detecting target analytes and enzymatic reactions
US7622294B2 (en) 1997-03-14 2009-11-24 Trustees Of Tufts College Methods for detecting target analytes and enzymatic reactions
US6406845B1 (en) 1997-05-05 2002-06-18 Trustees Of Tuft College Fiber optic biosensor for selectively detecting oligonucleotide species in a mixed fluid sample
AU756945B2 (en) * 1997-05-23 2003-01-30 Bioarray Solutions Ltd Color-encoding and in-situ interrogation of matrix-coupled chemical compounds
US7348181B2 (en) 1997-10-06 2008-03-25 Trustees Of Tufts College Self-encoding sensor with microspheres
US7115884B1 (en) * 1997-10-06 2006-10-03 Trustees Of Tufts College Self-encoding fiber optic sensor
US6265163B1 (en) 1998-01-09 2001-07-24 Lynx Therapeutics, Inc. Solid phase selection of differentially expressed genes
US6210910B1 (en) 1998-03-02 2001-04-03 Trustees Of Tufts College Optical fiber biosensor array comprising cell populations confined to microcavities
EP2360271A1 (en) 1998-06-24 2011-08-24 Illumina, Inc. Decoding of array sensors with microspheres
US6429027B1 (en) 1998-12-28 2002-08-06 Illumina, Inc. Composite arrays utilizing microspheres
US7510841B2 (en) * 1998-12-28 2009-03-31 Illumina, Inc. Methods of making and using composite arrays for the detection of a plurality of target analytes
US6846460B1 (en) * 1999-01-29 2005-01-25 Illumina, Inc. Apparatus and method for separation of liquid phases of different density and for fluorous phase organic syntheses
ATE469699T1 (en) * 1999-02-23 2010-06-15 Caliper Life Sciences Inc MANIPULATION OF MICROPARTICLES IN MICROFLUID SYSTEMS
US20060275782A1 (en) 1999-04-20 2006-12-07 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
US20030207295A1 (en) * 1999-04-20 2003-11-06 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US6544732B1 (en) * 1999-05-20 2003-04-08 Illumina, Inc. Encoding and decoding of array sensors utilizing nanocrystals
US6620584B1 (en) 1999-05-20 2003-09-16 Illumina Combinatorial decoding of random nucleic acid arrays
US8481268B2 (en) 1999-05-21 2013-07-09 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
US8080380B2 (en) * 1999-05-21 2011-12-20 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
US7604996B1 (en) 1999-08-18 2009-10-20 Illumina, Inc. Compositions and methods for preparing oligonucleotide solutions
EP1212599A2 (en) 1999-08-30 2002-06-12 Illumina, Inc. Methods for improving signal detection from an array
US7167615B1 (en) 1999-11-05 2007-01-23 Board Of Regents, The University Of Texas System Resonant waveguide-grating filters and sensors and methods for making and using same
US7955794B2 (en) * 2000-09-21 2011-06-07 Illumina, Inc. Multiplex nucleic acid reactions
US7361488B2 (en) * 2000-02-07 2008-04-22 Illumina, Inc. Nucleic acid detection methods using universal priming
US6913884B2 (en) * 2001-08-16 2005-07-05 Illumina, Inc. Compositions and methods for repetitive use of genomic DNA
WO2001057268A2 (en) 2000-02-07 2001-08-09 Illumina, Inc. Nucleic acid detection methods using universal priming
US8076063B2 (en) 2000-02-07 2011-12-13 Illumina, Inc. Multiplexed methylation detection methods
US20050214825A1 (en) * 2000-02-07 2005-09-29 John Stuelpnagel Multiplex sample analysis on universal arrays
US7582420B2 (en) 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
US7611869B2 (en) * 2000-02-07 2009-11-03 Illumina, Inc. Multiplexed methylation detection methods
US20020006617A1 (en) 2000-02-07 2002-01-17 Jian-Bing Fan Nucleic acid detection methods using universal priming
CA2399908A1 (en) * 2000-02-10 2001-08-16 Todd Dickinson Array of individual arrays as substrate for bead-based simultaneous processing of samples and manufacturing method therefor
US6770441B2 (en) 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same
EP1967595A3 (en) 2000-02-16 2008-12-03 Illumina, Inc. Parallel genotyping of multiple patient samples
ES2259666T3 (en) 2000-06-21 2006-10-16 Bioarray Solutions Ltd MOLECULAR ANALYSIS OF MULTIPLE ANALYTICS USING SERIES OF RANDOM PARTICLES WITH APPLICATION SPECIFICITY.
US9709559B2 (en) 2000-06-21 2017-07-18 Bioarray Solutions, Ltd. Multianalyte molecular analysis using application-specific random particle arrays
US7057704B2 (en) * 2000-09-17 2006-06-06 Bioarray Solutions Ltd. System and method for programmable illumination pattern generation
US20030045005A1 (en) * 2000-10-17 2003-03-06 Michael Seul Light-controlled electrokinetic assembly of particles near surfaces
US20040018491A1 (en) * 2000-10-26 2004-01-29 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
WO2002057743A2 (en) * 2001-01-17 2002-07-25 Streamline Proteomics, Llc. Methods of analyzing and sorting one or more analytes
US6790672B2 (en) * 2001-02-19 2004-09-14 Board Of Regents The University Of Texas System Encoded molecular sieve particle-based sensors
US7262063B2 (en) 2001-06-21 2007-08-28 Bio Array Solutions, Ltd. Directed assembly of functional heterostructures
US20030059850A1 (en) * 2001-09-26 2003-03-27 Psychiatric Genomics, Inc. Fluorescence proximity assay
US20050164261A1 (en) * 2001-10-09 2005-07-28 Chandler Don J. Multiplexed analysis of clinical specimens apparatus and methods
US8148171B2 (en) 2001-10-09 2012-04-03 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and methods
CA2741049C (en) 2001-10-15 2019-02-05 Bioarray Solutions, Ltd. Multiplexed analysis of polymorphic loci by probe elongation-mediated detection
US6838289B2 (en) 2001-11-14 2005-01-04 Beckman Coulter, Inc. Analyte detection system
DE10156612A1 (en) * 2001-11-20 2003-05-28 Markus Klotz To determine the presence of a substance, in a liquid or gas sample, a possible micro-particle size category is taken to be bonded with the sample substance by hybridizing for measurement by particle size/fluorescent light signals
CN100360722C (en) 2001-11-21 2008-01-09 艾普勒拉公司 Digital assay
US20030109067A1 (en) 2001-12-06 2003-06-12 Immunetech, Inc. Homogeneous immunoassays for multiple allergens
US7674632B1 (en) * 2001-12-10 2010-03-09 Zeus Scientific, Inc Method and composition for homogeneous multiplexed microparticle-based assay
WO2003069333A1 (en) 2002-02-14 2003-08-21 Illumina, Inc. Automated information processing in randomly ordered arrays
US7872804B2 (en) 2002-08-20 2011-01-18 Illumina, Inc. Encoded particle having a grating with variations in the refractive index
US7441703B2 (en) 2002-08-20 2008-10-28 Illumina, Inc. Optical reader for diffraction grating-based encoded optical identification elements
US7619819B2 (en) * 2002-08-20 2009-11-17 Illumina, Inc. Method and apparatus for drug product tracking using encoded optical identification elements
US7164533B2 (en) 2003-01-22 2007-01-16 Cyvera Corporation Hybrid random bead/chip based microarray
US7923260B2 (en) * 2002-08-20 2011-04-12 Illumina, Inc. Method of reading encoded particles
US7508608B2 (en) 2004-11-17 2009-03-24 Illumina, Inc. Lithographically fabricated holographic optical identification element
US7901630B2 (en) 2002-08-20 2011-03-08 Illumina, Inc. Diffraction grating-based encoded microparticle assay stick
US7900836B2 (en) 2002-08-20 2011-03-08 Illumina, Inc. Optical reader system for substrates having an optically readable code
AU2003298412A1 (en) * 2002-09-03 2004-03-29 Zeptosens Ag Analytical platform and identification method
AU2003267192A1 (en) 2002-09-12 2004-04-30 Cyvera Corporation Method and apparatus for aligning elongated microbeads in order to interrogate the same
US20100255603A9 (en) 2002-09-12 2010-10-07 Putnam Martin A Method and apparatus for aligning microbeads in order to interrogate the same
US7092160B2 (en) 2002-09-12 2006-08-15 Illumina, Inc. Method of manufacturing of diffraction grating-based optical identification element
US20040058333A1 (en) * 2002-09-23 2004-03-25 Bell Michael L. Assay products and procedures
US20040259105A1 (en) * 2002-10-03 2004-12-23 Jian-Bing Fan Multiplex nucleic acid analysis using archived or fixed samples
US7526114B2 (en) 2002-11-15 2009-04-28 Bioarray Solutions Ltd. Analysis, secure access to, and transmission of array images
US7326573B2 (en) * 2003-01-10 2008-02-05 Beckman Coulter, Inc. Assay procedures and apparatus
US6943768B2 (en) 2003-02-21 2005-09-13 Xtellus Inc. Thermal control system for liquid crystal cell
WO2005029705A2 (en) 2003-09-18 2005-03-31 Bioarray Solutions, Ltd. Number coding for identification of subtypes of coded types of solid phase carriers
ES2375962T3 (en) 2003-09-22 2012-03-07 Bioarray Solutions Ltd IMMOBILIZED SURFACE POLYELECTROLYTE WITH MULTIPLE FUNCTIONAL GROUPS ABLE TO JOIN COVALENTLY TO BIOMOLECULES.
CA2899287A1 (en) 2003-10-28 2005-05-12 Bioarray Solutions Ltd. Optimization of gene expression analysis using immobilized capture probes
AU2004287069B2 (en) 2003-10-29 2009-07-16 Bioarray Solutions, Ltd. Multiplexed nucleic acid analysis by fragmentation of double-stranded DNA
US7323696B2 (en) 2004-01-09 2008-01-29 Applera Corporation Phosphor particle coded beads
US7433123B2 (en) 2004-02-19 2008-10-07 Illumina, Inc. Optical identification element having non-waveguide photosensitive substrate with diffraction grating therein
US7848889B2 (en) 2004-08-02 2010-12-07 Bioarray Solutions, Ltd. Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification
ATE459933T1 (en) 2004-11-16 2010-03-15 Illumina Inc METHOD AND APPARATUS FOR READING CODED MICROBALLS
US7604173B2 (en) 2004-11-16 2009-10-20 Illumina, Inc. Holographically encoded elements for microarray and other tagging labeling applications, and method and apparatus for making and reading the same
WO2006055735A2 (en) 2004-11-16 2006-05-26 Illumina, Inc Scanner having spatial light modulator
CA2589195A1 (en) * 2004-11-29 2006-06-01 Perkinelmer Las, Inc. Particle-based multiplex assay for identifying glycosylation
US20060246576A1 (en) 2005-04-06 2006-11-02 Affymetrix, Inc. Fluidic system and method for processing biological microarrays in personal instrumentation
US8486629B2 (en) 2005-06-01 2013-07-16 Bioarray Solutions, Ltd. Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation
US20070043510A1 (en) * 2005-08-19 2007-02-22 Beckman Coulter, Inc. Assay system
US7623624B2 (en) 2005-11-22 2009-11-24 Illumina, Inc. Method and apparatus for labeling using optical identification elements characterized by X-ray diffraction
US20090104613A1 (en) * 2005-12-23 2009-04-23 Perkinelmer Las, Inc. Methods and compositions relating to multiplexed genomic gain and loss assays
MX2008008079A (en) 2005-12-23 2008-10-22 Perkinelmer Las Inc Comparative genomic hybridization on encoded multiplex particles.
EP1969337A4 (en) * 2005-12-23 2010-01-27 Perkinelmer Las Inc Multiplex assays using magnetic and non-magnetic particles
US20100009373A1 (en) * 2005-12-23 2010-01-14 Perkinelmer Health Sciences, Inc. Methods and compositions relating to multiplex genomic gain and loss assays
WO2007075910A2 (en) * 2005-12-23 2007-07-05 Perkinelmer Las, Inc. Methods and compositions for detecting enzymatic activity
US7932037B2 (en) * 2007-12-05 2011-04-26 Perkinelmer Health Sciences, Inc. DNA assays using amplicon probes on encoded particles
US7830575B2 (en) 2006-04-10 2010-11-09 Illumina, Inc. Optical scanner with improved scan time
WO2007127988A2 (en) * 2006-04-28 2007-11-08 Perkinelmer Las, Inc. Detecting phospho-transfer activity
WO2008097371A2 (en) * 2006-10-02 2008-08-14 Agave Biosystems, Inc. Modular microfluidic flow cytometer and method applications
US9146233B2 (en) * 2006-11-13 2015-09-29 Perkinelmer Health Sciences, Inc. Detecting molecular interactions by fluorescence resonance energy transfer on a solid-phase support
US8288110B2 (en) * 2006-12-04 2012-10-16 Perkinelmer Health Sciences, Inc. Biomarkers for detecting cancer
US9164037B2 (en) * 2007-01-26 2015-10-20 Palo Alto Research Center Incorporated Method and system for evaluation of signals received from spatially modulated excitation and emission to accurately determine particle positions and distances
US8821799B2 (en) 2007-01-26 2014-09-02 Palo Alto Research Center Incorporated Method and system implementing spatially modulated excitation or emission for particle characterization with enhanced sensitivity
US20080279874A1 (en) * 2007-05-07 2008-11-13 Wyeth Compositions and methods for modulation of plk1 kinase activity
BRPI0812461A2 (en) 2007-06-08 2017-06-13 Biogen Idec Inc method of predicting an individual's response to therapy, method of treating an immune disorder, composition and kit
US8629981B2 (en) * 2008-02-01 2014-01-14 Palo Alto Research Center Incorporated Analyzers with time variation based on color-coded spatial modulation
US9523701B2 (en) 2009-07-29 2016-12-20 Dynex Technologies, Inc. Sample plate systems and methods
GB0913258D0 (en) 2009-07-29 2009-09-02 Dynex Technologies Inc Reagent dispenser
US20110046009A1 (en) * 2009-08-24 2011-02-24 Perkinelmer Health Sciences, Inc. Methods for detecting dna methylation using encoded particles
CA2774116A1 (en) 2009-09-25 2011-03-31 Signature Genomics Laboratories Llc Multiplex (+/-) stranded arrays and assays for detecting chromosomal abnormalities associated with cancer and other diseases
US10273544B2 (en) 2010-02-11 2019-04-30 Dana-Farber Cancer Institute, Inc. Methods for predicting likelihood of responding to treatment
JP2012180049A (en) 2011-03-02 2012-09-20 Toyota Industries Corp Air-conditioning system for vehicle
US9029800B2 (en) 2011-08-09 2015-05-12 Palo Alto Research Center Incorporated Compact analyzer with spatial modulation and multiple intensity modulated excitation sources
US8723140B2 (en) * 2011-08-09 2014-05-13 Palo Alto Research Center Incorporated Particle analyzer with spatial modulation and long lifetime bioprobes
US20180119221A1 (en) 2015-04-02 2018-05-03 Hmnc Value Gmbh Method of Treatment Using Genetic Predictors of a Response to Treatment with CRHR1 Antagonists
ES2724100T3 (en) 2015-04-02 2019-09-06 HMNC Diagnostics GmbH Genetic prediction factors of a response to treatment with CRHR1 antagonists
CN108507983A (en) * 2017-02-24 2018-09-07 深圳市液芯生物科技有限公司 A kind of coding/decoding method and device of fluorescence-encoded micro-beads
US11408885B2 (en) 2017-08-31 2022-08-09 Massachusetts Institute Of Technology Compositions and multiplex assays for characterizing active proteases and their inhibitors
KR20220098146A (en) * 2019-10-17 2022-07-11 씨2센스, 인크. Luminescence imaging for detection
EP4283299A3 (en) 2020-04-14 2024-02-28 (INSERM) Institut National de la Santé et de la Recherche Médicale Methods for detecting the presence of coronavirus-specific antibodies in a subject
WO2022043415A1 (en) 2020-08-27 2022-03-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for detecting the presence of pemphigus-specific autoantibodies in a sample

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1980002076A1 (en) * 1979-03-19 1980-10-02 Diagnostic Techn Int Inc Double tagged immunoassay
GB2126341A (en) * 1982-08-30 1984-03-21 Becton Dickinson Co Method and apparatus for distinguishing subpopulations of cells
EP0121262A2 (en) * 1983-04-05 1984-10-10 Becton Dickinson and Company Method and apparatus for distinguishing multiple subpopulations of cells in a sample
EP0126450A2 (en) * 1983-05-19 1984-11-28 Ioannis Dr. Tripatzis Particle and method for the detection of antigens and/or antibodies using this particle
US4584277A (en) * 1983-04-05 1986-04-22 Syntex (U.S.A.) Inc. Fluorescent multiparameter particle analysis

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4727020A (en) * 1985-02-25 1988-02-23 Becton, Dickinson And Company Method for analysis of subpopulations of blood cells
JPS6281566A (en) * 1985-10-07 1987-04-15 Showa Denko Kk Quantification method by measurement of fluorescent intensity of fine particle
JP3496588B2 (en) * 1999-09-14 2004-02-16 ダイキン工業株式会社 Air purifier and its ionization unit

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1980002076A1 (en) * 1979-03-19 1980-10-02 Diagnostic Techn Int Inc Double tagged immunoassay
GB2126341A (en) * 1982-08-30 1984-03-21 Becton Dickinson Co Method and apparatus for distinguishing subpopulations of cells
EP0121262A2 (en) * 1983-04-05 1984-10-10 Becton Dickinson and Company Method and apparatus for distinguishing multiple subpopulations of cells in a sample
US4584277A (en) * 1983-04-05 1986-04-22 Syntex (U.S.A.) Inc. Fluorescent multiparameter particle analysis
EP0126450A2 (en) * 1983-05-19 1984-11-28 Ioannis Dr. Tripatzis Particle and method for the detection of antigens and/or antibodies using this particle

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Nucleic Acids Research, Vol. 14, No 2, 1986, pages 1017-1028 (A.-C. SYVANEN et al) "Time-Resolved Fluorometry: a Sensitive Method to Quantify DNA-Hybrids" * whole document * *

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0446245B1 (en) * 1988-11-03 1999-05-12 Igen International, Inc. Electrochemiluminescent assays
WO1991012530A1 (en) * 1990-02-16 1991-08-22 Pa Consulting Services Limited Radiative transfer fluorescence assay
WO1993003379A1 (en) * 1991-07-26 1993-02-18 E.I. Du Pont De Nemours And Company An assay with signal detection in the presence of a suspended solid support
EP0531933A2 (en) * 1991-09-09 1993-03-17 Canon Kabushiki Kaisha Quantitative determination of liquids, pure or complexed with other components
US5770452A (en) * 1991-09-09 1998-06-23 Canon Kabushiki Kaisha Quantitative determination of lipid, and of co-existing two compounds
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EP0563998A1 (en) * 1992-04-02 1993-10-06 Markus Sauer A process for detecting biomolecules, toxic substances, polymers and pharmaceutical substances by time-resolved laser spectroscopy
US5518883A (en) * 1992-07-02 1996-05-21 Soini; Erkki J. Biospecific multiparameter assay method
WO1994001774A1 (en) * 1992-07-02 1994-01-20 Erkki Soini Biospecific multiparameter assay method
EP0617287A3 (en) * 1993-03-16 1995-07-26 Wallac Oy Biospecific assay method.
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US5879894A (en) * 1993-03-19 1999-03-09 Chiron Diagnostics Corporation Long emission wavelength chemiluminescent compounds and their use in test assays
US5702887A (en) * 1993-03-19 1997-12-30 Chiron Diagnostics Corporation Long emission wavelength chemiluminescent compounds and their use in test assays
WO1996031777A1 (en) * 1995-04-03 1996-10-10 Macquarie Research Limited Method for detecting microorganisms
US6225046B1 (en) 1995-04-03 2001-05-01 Macquarie Research Ltd. Method for detecting microorganisms
FR2735577A1 (en) * 1995-06-15 1996-12-20 Inst Francais Du Petrole METHOD AND DEVICE FOR EVALUATING THE PROGRESS OF A TRANSIENT PHENOMENON
EP0749012A1 (en) * 1995-06-15 1996-12-18 Institut Français du Pétrole Device and method for determining the temporal behaviour of a transient phenomenon
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US6342397B1 (en) * 1998-06-04 2002-01-29 Erkki Soini Homogeneous biospecific assay using a solid phase, two-photon excitation and confocal fluorescence detection
US6979830B2 (en) 1998-08-21 2005-12-27 Ppd Biomarker Discovery Sciences, Llc Optical architectures for microvolume laser-scanning cytometers
EP1121582A2 (en) * 1998-08-21 2001-08-08 SurroMed, Inc. Novel optical architectures for microvolume laser-scanning cytometers
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US6603537B1 (en) 1998-08-21 2003-08-05 Surromed, Inc. Optical architectures for microvolume laser-scanning cytometers
US6800860B2 (en) 1998-08-21 2004-10-05 Surromed, Inc. Optical architectures for microvolume laser-scanning cytometers
WO2000012123A2 (en) * 1998-08-28 2000-03-09 Febit Ferrarius Biotechnology Gmbh Method and measuring device for determining a plurality of analytes in a sample
WO2000012123A3 (en) * 1998-08-28 2001-04-12 Febit Ferrarius Biotech Gmbh Method and measuring device for determining a plurality of analytes in a sample
WO2002027300A1 (en) * 2000-09-27 2002-04-04 Amersham Biosciences Uk Ltd Measurement of fluorescence decay times
US6809816B2 (en) 2000-09-27 2004-10-26 Amersham Biosciences Uk Limited Measurement of fluorescence decay times
WO2002061427A1 (en) * 2001-01-31 2002-08-08 Novozymes A/S Method of analysing granular composition by fluorescence analysis
WO2004106931A1 (en) * 2003-05-26 2004-12-09 Institut Virion\Serion Gmbh Method and testing system for analyzing and/or detecting biomolecules and/or active substances in liquid samples
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SE458968B (en) 1989-05-22
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